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THE decrease in vascular tone observed during isoflurane anesthesia has been related in part to an alteration of the vascular smooth muscle cells’ (VSMC) vasomotor response to vasoactive substances, including norepinephrine, endothelin, or vasopressin. 1–3 Angiotensin II (AngII) is a potent vasoconstrictor involved in the short-term control of arterial blood pressure during isoflurane anesthesia. 4 The hemodynamic effects of AngII are mediated through binding to angiotensin subtype 1 (AT1) receptors, which belong to the G protein– coupled receptor family. Activation of AT1receptor stimulates phospholipase C to hydrolyze phophatidylinositol 4,5-biphosphate, thereby producing inositol 1,4,5-triphosphate. The latter then binds to its specific receptor located on Ca2+stores to release Ca2+from internal stores, and to activate Ca2+influx from extracellular spaces. 5–7 A normal response of VSMC to AngII may be critical for hemodynamic stability in several conditions encountered during the perioperative period, especially hypovolemia. However, the effect of isoflurane on AngII response is not described.

The present study was undertaken to assess the effect of isoflurane on AngII-induced intracellular Ca2+mobilization and to gain further insight into the cellular mechanisms by which isoflurane could modulate AngII signaling. One mechanism that has been associated with intracellular Ca2+mobilization is cytoskeletal function. We were particularly interested in the effect of isoflurane on microtubular and actin networks because several volatile anesthetic agents have been reported to directly interact with the organization of cytoskeletal elements, 8,9 and both actin and microtubular networks were recently reported to be involved in the transduction of several extracellular signals, including G protein–coupled transduction pathways and Ca2+mobilization from internal store, or cell contraction in response to various agonists. 10–16

On the other hand, structural and functional abnormalities of the arterial wall have been reported in some experimental models of genetic hypertension and have been associated with an increase in intracellular Ca2+mobilization in VSMC in response to several agonists, especially AngII. 17–23 These abnormalities could partly explain the lability of blood pressure observed during general anesthesia in individuals who have essential hypertension. 24,25 For this purpose we compared the effects of isoflurane in VSMC from spontaneously hypertensive rats (SHR) to normotensive control (Wistar Kyoto rats [WKY] ).

Material and Methods

Cell Culture

The study was approved by the Institutional Animal Investigation Committee and was conducted following recommendations established by the European Community Guidelines for Animal Ethical Care and Use of Laboratory Animals (Directive 86/609). Young male WKY/NIco rats (mean arterial pressure ± SEM = 98 ± 4 mmHg; n = 30) and SHR/NIco (mean arterial pressure ± SEM = 136 ± 5 mmHg; n = 25) aged 6 weeks were obtained from Iffa-Credo (L'Arbresle, France) and were used throughout the study. Cultured VSMC were obtained by enzymatic digestion as previously described. 16,26 In brief, aortas were incubated for 10 min in Dulbecco's modified Eagle medium (Eurobio, Les Ulis, France) supplemented with glutamine (2 mm), 0.1% bovine serum albumin, penicillin (10 U/ml), streptomycin (100 mg/ml), and collagenase (295 U/ml). After mechanical removal of adventitial and endothelial cell layers, the media was incubated in dissecting solution (20 min, 37°C) to which elastase (90 U/ml) and pronase (0.33 mg/ml) had been added. Cells were then detached by gentle pipetting of the tissue through a large hole Pasteur pipette. The procedure was then repeated twice on undigested tissue.

Isolated cells were seeded at 1.5–2.0 × 105cells/ml into 25-cm2flasks in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (Eurobio), 2 mm l-glutamine, 25 mm HEPES, pH 7.4, 10 U/ml penicillin, and 100 mg/ml streptomycin, and incubated at 37°C and 5% CO2in a humidified incubator. At confluence, secondary cultures were obtained by serial passage after the cells were harvested with 0.5 g/l trypsin and 0.2 g/l EDTA (Sigma-Aldrich, Saint Quentin Falavier, France) and reseeded in fresh Dulbecco's modified Eagle medium containing 10% fetal calf serum. Cells between the third and ninth passage were studied at confluence and were made quiescent by incubation for 48 h in a fetal calf serum–free media (0.5%) before the experiment. At least 10 sets of cells were analyzed at each passage.

Cell Ca2+Measurements

Intracellular Ca2+variations were assessed in single cells using fluorescence imaging as described previously. 16 In brief, cells were loaded with the acetoxymethyl ester form of Fura-2 (5 μm, 30 min at 37°C; Molecular Probe, AA Leiden, The Netherlands). The coverslips were mounted on the stage of a Nikon Diaphot microscope (Nikon, Kawasaki, Japan) fitted with a cooled integrating Coupled Charged Device imaging system (Newcastle Photometric System, Newcastle upon Tyne, United Kingdom), and cells were superfused with Na+–HEPES solution at 37°C at a flow rate of 1 ml/min. An area on the coverslip chamber containing 10–15 cells was randomly selected with the microscope, and the position of the objective was focused to view the median section of the cells. Cells were illuminated alternately at 350 and 380 nm, and the intensity of emitted light from single cells during a 500-ms period at wavelength greater than 520 nm was measured. The ratio of the light intensities at the two wavelengths, plotted against time, was used to reflect qualitative changes in intracellular Ca2+. 27 Because calibration procedures are prone to error, no attempt was made to calibrate the ratio values. 5,28,29

Staining of Filamentous Actin and α-Tubulin

To assess the effect of isoflurane on the organization of the cytoskeleton, the actin and microtubular networks were visualized using double indirect immunofluorescence labeling. After two washes in phosphate-buffered saline (Life Technologies, Cergy-Pontoise, France), VSMC were fixed in 4% formaldehyde for 10 min and then permeabilized 10 min with 0.2% Triton X-100 (Sigma-Aldrich, Saint Quentin Falavier, France) at room temperature. Cultures were incubated in phosphate-buffered saline containing 5% bovine serum albumin for 60 min to block nonspecific binding sites. This was followed by overnight incubation at 4°C in a humidified chamber in the presence of monoclonal antibodies directed against α-tubulin (1/200; Sigma-Aldrich) in phosphate-buffered saline containing 2% bovine serum albumin. After washing, horse biotinylated antimouse immunoglobulin G (1/30; Vector Laboratories, Abcys, Paris, France) was added for 60 min at room temperature. Cells were washed again and incubated for 60 min with Streptavidin-Texas red (1/30; Amersham, Bioscience, Orsay, France). This was followed after washes by an incubation with fluorescein isothiocyanate conjugated–phalloidin (1/20; Sigma-Aldrich) for 90 min. After a final wash, coverslips were placed in mounting medium (Fluoprep; Merieux, Lyon, France). Slides were examined with a Leica DMLB fluorescence microscope set for fluorescein and Texas red fluorescence and connected to a Sony 3 CCD DXC 930P color camera (Sony Corp., Tokyo, Japan). The resulting images were printed on an Epson Stylus Photo 890 printer (Seiko Epson Corp., Nagano, Japan). In control experiments, the incubation steps with primary antibodies were omitted. Experiments were performed in triplicate.

Disorganization of Actin or Tubular Networks

A common approach to assess the implication of cytoskeletal elements in cellular response is to hamper the organization of a network using specific agents. Preliminary experiments were conducted to determine the optimal concentration and time of incubation for each agent to impair network organization without inducing cell shape modification. Cells were incubated 30 min at 37°C in the presence of 5 μm nocodazole, 10 μm vinblastine, 8 μm paclitaxel or 2 μm cytochalasin D and then either stimulated by AngII to assess intracellular Ca2+mobilization or fixed for tubulin and actin immunostaining.

Experimental Procedure

Cells were exposed to AngII, which induced a rapid increase in intracellular Ca2+(figs. 1A–D) followed by a return toward initial values. Ca2+transient was characterized by its amplitude (peak minus basal ratio) and slope of intracellular Ca2+increase. The optimal concentration of AngII that induced a maximal mobilization of Ca2+in both stains was determined by increasing concentration (0.1 nm to 10.0 μ m) in both WKY and SHR strains. 16 In accordance with previous studies, this was observed for concentrations greater than 0.5 μm. 19 Subsequently, the effect of AngII was assessed at 1 μm in both strains. AT1antagonists CGP-48 933 (100 nm) and CI-996 (100 nm) inhibited the response to AngII in both strains (> 95% inhibition, P
< 0.0001 for each, results not shown). AngII-induced intracellular Ca2+mobilization was not significantly different in cells from the third to the ninth passage in both strains (F = 0.26, analysis of variance;P
= not significant [NS]). As previously described, AngII induced a receptor desensitization that precluded repetitive stimulation of the same cell by AngII. 30

Fig. 1. Representative recordings of the effect of 1 μm angiotensin II (AngII) on intracellular Ca2+increase in a control cell from WKY and in cells incubated in the presence of 1, 2, or 3% isoflurane. In protocols A
–D
, cells were exposed to AngII in the presence of external Ca2+(Cao2+). In protocols E
–H
, cells were incubated for 60 s in the nominal absence of external Ca2+and then exposed to Ang II. External Ca2+was reintroduced in the perfusion medium and AngII-induced Ca2+influx was estimated by the time course of Ca2+increase. In recordings B
–D
and F
–H
, cells were incubated in Na+–HEPES medium containing 1, 2, and 3% isoflurane, respectively (15 min). Ratios of the emission fluorescence (> 520 nm) measured at excitation wavelengths of 350 and 380 nm are shown on the ordinate.

Fig. 1. Representative recordings of the effect of 1 μm angiotensin II (AngII) on intracellular Ca2+increase in a control cell from WKY and in cells incubated in the presence of 1, 2, or 3% isoflurane. In protocols A
–D
, cells were exposed to AngII in the presence of external Ca2+(Cao2+). In protocols E
–H
, cells were incubated for 60 s in the nominal absence of external Ca2+and then exposed to Ang II. External Ca2+was reintroduced in the perfusion medium and AngII-induced Ca2+influx was estimated by the time course of Ca2+increase. In recordings B
–D
and F
–H
, cells were incubated in Na+–HEPES medium containing 1, 2, and 3% isoflurane, respectively (15 min). Ratios of the emission fluorescence (> 520 nm) measured at excitation wavelengths of 350 and 380 nm are shown on the ordinate.

AngII-induced Ca2+mobilization from internal stores was assessed in the absence of external Ca2+(figs. 1E– H). Ca2+transient was characterized by its amplitude (peak minus basal ratio), slope of intracellular Ca2+increase, and total Ca2+released, estimated by area under transient. As previously described, Ca2+mobilization was higher in the SHR than in WKY (table 1). 16,19

AngII-induced Ca2+influx was estimated after exposure of cells to AngII in the absence of external Ca2+, from the rate of intracellular Ca2+increase following reintroduction of external Ca2+(fig. 1E). In preliminary experiments, intracellular Ca2+changes occurring in nonstimulated cells as a consequence of Ca2+chelation and reintroduction were found to be very low, with a mean of 0.002 ratio unit/min. Ca2+influx elicited by AngII was not significantly altered by nifedipine (5 μm) in either strain (WKY: 98 ± 8% of control values, n = 73; SHR: 93 ± 14% of control values, n = 43;P
= NS for each). To assess the role of the Na+– Ca2+exchanger in AngII-induced Ca2+influx, experiments were performed in the absence of external Na+, which had been replaced by N
-methyl-glucamine (NMG; Sigma-Aldrich). Ca2+influx was similar in Na+–HEPES and NMG–HEPES solution in both WKY (Na+–HEPES: 0.396 ± 0.024 ratio unit/min; NMG–HEPES: 0.402 ± 0.042; n = 109;P
= NS) and SHR (Na+–HEPES: 0.481 ± 0.048 ratio unit/min; NMG– HEPES: 0.410 ± 0.040; n = 103;P
= NS). This suggests that the participation of the Na+– Ca2+exchanger in Ca2+influx is negligible in our conditions.

Anesthetic Agent

Output from an isoflurane calibrated vaporizer (Fortec 3; Cyprane LTD, Keighley, United Kingdom) was bubbled through 100-ml glass bottles containing either Na+–HEPES or Ca2+-free Na+–HEPES solution and allowed to equilibrate for 60 min at 37°C. Air at a flow rate of 1 l/min was used as a carrier gas. Isoflurane (obtained from Abbott Laboratories, Cergy, France) was studied at concentration of 0.5, 1, 2, or 3 vol% in air. No correction was performed to adjust for interspecies variation of anesthetic potency because end-tidal isoflurane requirement to achieve a 1.0 minimal alveolar concentration level of anesthesia has been reported to be very similar in WKY (mean ± SD: 1.20 ± 0.05%) and SHR (mean ± SD: 1.26 ± 0.07; difference NS). 31 Teflon tubing was used to reduce anesthetic agent loss between the bottle and the recording chamber. Aqueous phase concentrations of isoflurane in the solution, sampled at the recording chamber level, were determined by direct spectrometry at three wavelengths (210, 280, and 600 nm) using a variable wavelength spectrometer (model 1050; Hewlett Packard, Les Ulis, France). 32 Isoflurane concentrations measured after 60-min equilibration with the superfusion solution were 0.27 ± 0.05, 0.51 ± 0.08, and 0.68 ± 0.13 mm (mean ± SEM) at 1, 2, and 3%, respectively. To ensure stable isoflurane concentration in the solution at the recording chamber level, the chamber itself was covered by a semiclosed Plexiglass reservoir, continuously aerated by a derivation from the output of the vaporizer. Isoflurane concentration in the gas phase of this reservoir was continuously monitored by an infrared calibrated analyzer (Capnomac; Datex Instrumentarium, Helsinki, Finland).

Results are presented as mean ± SEM. The values of parameters of Ca2+mobilization measured in treated cells are expressed as percentage of the control values (untreated cells). Student t
test for unpaired data were used to compare mean values obtained in control cells with values obtained in treated cells and to compare mean values obtained in WKY with those in SHR. A comparison of the values obtained in cells from passages 3–9 for each strain was performed by analysis of variance with multiple testing according to the Bonferroni method. Statistical analysis were performed using Statview 5.1 software (SAS Institute Inc., Cary, NC). P
< 0.05 was considered significant.

To assess the effect of isoflurane on total intracellular Ca2+mobilization, cells loaded with Fura-2 were incubated in the presence of 0.5–3% isoflurane for 15 min and then exposed to AngII in the presence of external Ca2+. In both strains, isoflurane induced a concentration-dependent decrease in intracellular Ca2+release elicited by AngII in the presence of external Ca2+(WKY:figs. 1A–Dand table 2; SHR:table 3). This effect was observed at concentration of isoflurane greater than 0.5% for WKY and 1% for SHR.

To assess the effect of isoflurane on AngII-induced intracellular Ca2+release from internal stores, cells were incubated in the presence of 0.5–3% isoflurane for 15 min and then exposed to AngII in Ca2+-free medium. Ca2+influx was assessed on reintroduction of external Ca2+. In both strains, isoflurane decreased Ca2+release from internal stores and Ca2+influx in a concentration-dependent manner (WKY:figs. 1E–Hand table 2; SHR:table 3).

To assess the effect of isoflurane on Ca2+stores operated channels, a depletion of Ca2+intracellular stores was induced by exposing cells to thapsigargin (3 μm), an inhibitor of the sarco/endoplasmic reticulum Ca2+adenosine triphosphatase, in the absence of external Ca2+. In both strains, this induced a transient intracellular Ca2+increase at a slower rate than that observed with AngII. Incubation of VSMC with thapsigargin for 5 min abolished the response to subsequent infusion of AngII in the two strains (results not shown), as previously reported. 19 Furthermore, thapsigargin completely depleted intracellular Ca2+stores because ionomycin addition did not elicit any increase in cell Ca2+(WKY: 98 ± 1% of inhibition, n = 28; SHR: 98 ± 2% of inhibition, n = 25;P
< 0.001 for each). Reintroduction of Ca2+(1 mm) into the medium induced a Ca2+influx of similar magnitude to that observed after stimulation with 1 μm AngII. Isoflurane at concentration of 1–3% and 2–3% significantly decreased thapsigargin-induced Ca2+influx in WKY (table 2) and SHR (table 3), respectively.

Reversibility of Isoflurane Action

To assess the reversibility of the effect of isoflurane, cells were exposed to 3% isoflurane for 15 min and washed out with isoflurane-free Na+–HEPES solution for another 60-min period. In both strains, the effect of isoflurane on AngII-induced Ca2+mobilization (results not shown) was completely reversible 60 min after anesthetic retrieval.

Interaction between Cytoskeleton Elements and Isoflurane

Cytoskeletal elements have been shown recently to be involved in the Ca2+mobilization induced by AngII. 16 We tested the hypothesis that the inhibitory effect of isoflurane could be linked to an interaction between isoflurane and actin or tubulin networks.

Effect of Disorganizing Cytoskeleton Elements on Isoflurane Action

We first studied the effect of an alteration of cytoskeleton dynamic on the inhibition AngII-induced Ca2+mobilization by isoflurane.

To assess the implication of α-tubulin network, cells were incubated in the presence of nocodazole (5 μm, 30 min at 37°C) and exposed to 2% isoflurane. At this concentration, nocodazole disorganized microtubular network without altering cell shape (WKY:fig. 2, SHR:fig. 3), and, as expected, no differential effect could be observed on actin network with this agent (result not shown). As previously described, 16 pretreatment with nocodazole significantly reduced AngII-induced Ca2+mobilization from internal stores (fig. 4), whereas Ca2+influx was not significantly altered in both strains. In both WKY and SHR, 2% isoflurane had no significant effect on Ca2+mobilization in cells pretreated with nocodazole (fig. 4). Similar results on Ca2+release from internal stores were obtained with vinblastine or paclitaxel, which alter organization of microtubules by different mechanisms, in both WKY and SHR (fig. 4).

The role of actin network was assessed using pretreatment with cytochalasin D at a concentration of 2 μm for 30 min, known to disrupt actin network, with the formation of patches scattered throughout the cytoplasm, without altering cell shape (control:fig. 5A[WKY] and fig. 5B[SHR]; cytochalasin D:fig. 5C[WKY] and fig. 5D[SHR]). As previously described, 16 pretreatment with cytochalasin D significantly decreased the AngII-induced intracellular Ca2+transient in the SHR (amplitude: 61 ± 14% of control value;P
< 0.001), whereas it was not altered in cells from WKY (105 ± 8%;P
= NS). Pretreatment with cytochalasin D did not significantly alter the effect of 2% isoflurane in both WKY (44 ± 9% of control;P
< 0.01) and SHR (56 ± 8% of control;P
< 0.01).

We also tested the hypothesis that the effect of isoflurane on AngII-induced Ca2+mobilization could be linked to an alteration by isoflurane itself of cytoskeletal network organization. To study this, immunofluorescent staining of α-tubulin and actin were performed in VSMC exposed to 1, 2, and 3% isoflurane for 15 min. In control conditions, α-tubulin network was regular and homogeneous in both WKY (fig. 2A) and SHR (fig. 3A). Isoflurane altered in a dose-dependent manner the tubular organization with formation of α-tubulin aggregates. This effect was observed at a concentration of 1% or greater in the WKY and 2% or greater in SHR (figs. 2 and 3). It is noteworthy that at 3% isoflurane, alteration in α-tubulin networks were similar to that produced by nocodazole (5 μm;figs. 2B and 3B). These alterations were reversible 60 min after anesthetic retrieval (figs. 2F and 3F).

The current study demonstrated that clinically relevant concentrations of isoflurane decreased Ca2+mobilization elicited by AngII in both normotensive and hypertensive rat strains. Isoflurane reduced both AngII-induced Ca2+mobilization from internal stores and Ca2+influx through nifedipine-insensitive Ca2+channels. The hypertensive strain was found more resistant to isoflurane than the normotensive one. This effect appeared to be strongly associated with a disorganization of the microtubular network by the anesthetic agent in both strains.

The effect of isoflurane on AngII-induced Ca2+mobilization is consistent with the effect of isoflurane on Ca2+mobilization elicited by other agonists known to stimulate phospholipase C, such as vasopressin. 33 An effect of isoflurane on Ca2+stores per se
is unlikely, as no significant increase in cell Ca2+could be observed when cells were exposed to isoflurane alone. This is in accord with previous data reported by Akata et al.34 This suggests that the inhibition by isoflurane of AngII-induced Ca2+increase is related to an alteration in the dynamic of Ca2+release elicited by AngII, rather than an effect on Ca2+stores. This effect does not seem to be consequent to an interaction with the AT1receptors or AT1-related Gq11protein, because isoflurane did not affect AngII-induced inositol 1,4,5-triphosphate synthesis or lipophosphatidate signaling. 35,36

We therefore tested the hypothesis that isoflurane could affect the signaling pathway of AngII downstream of inositol 1,4,5-triphosphate synthesis. Our working hypothesis was that an interaction with microtubules network could be responsible of the inhibition of Ca2+release from internal stores. Previous studies have reported the role of microtubular network in the transduction of extracellular signal in various cell lines. In this regard, AngII-induced Ca2+mobilization in VSMC from both WKY and SHR strains was reported to require an intact microtubular network. 16 On the other hand, Allison et al.8 have shown, using electron microscopy, that short-term exposure of cells to halogenated anesthetics could induce dispersal of the microtubule system and disaggregation of the microtubules. 8 This effect was concentration-dependent and reversible after withdrawal of the agent and was found to be associated with a direct effect on microtubule assembly. 9 More recently, an intact microtubular network was shown to be critical in the process of isoflurane-induced preconditioning in cardiomyocytes. 37 Our results showed that the effect of isoflurane on AngII-induced Ca2+release was abolished in cells pretreated with either nocodazole, vinblastine, or paclitaxel, agents known to alter the micotubular dynamics by different mechanisms. These results, taken together with the direct and reversible effect of isoflurane on microtubular assembly, strongly suggest that the effect of isoflurane on Ca2+release may be related to an interaction with microtubular network, at a level that remains to be determined. This implication of the microtubules in the signaling pathways linking AngII receptor to Ca2+release appears to be similar in both WKY and SHR. In contrast, isoflurane had no visible effect on actin network, and disorganizing the actin network alone did not affect the response to isoflurane in the WKY.

Agonist-stimulated release of intracellular Ca2+from the intracellular stores is accompanied by repletion of the store by Ca2+influx from the extracellular space. 7 It is now recognized that the action of AngII in VSMC involves both voltage-operating channels and voltage-independent channels. 38 The relative contribution of these two pathways to cell Ca2+increase depends on the VSMC type and experimental conditions. 39 The lack of effect of nifedipine in this study suggests that Ca2+influx is mediated by voltage-independent channels, in accordance with previous reports. 6 This is further supported by our observation that agonist-independent depletion of intracellular Ca2+stores with thapsigargin activates a Ca2+entry pathway. This pathway, termed “capacitative Ca2+entry,” has been reported in different cell types, including VSMC, is insensitive to nifedipine, and contributes to smooth muscle tone. 39–41 The current results show that isoflurane inhibits both AngII- and thapsigargin-induced Ca2+influx. This is in accord with studies showing that isoflurane inhibited the Ca2+influx elicited by vasopressin or platelet-derived growth factor in A7r5 aortic VSMC and by bradykininin in bovine aortic endothelial cells. 33,42 In contrast, Hirata et al.43 have shown that isoflurane enhanced receptor-operated Ca2+influx in VSMC from rat thoracic aorta submaximally activated by phenylephrine. These discrepencies may be related to the diversity of influx pathways and the complexity of their regulation by signaling elements.

The increase in intracellular free Ca2+is the principal mechanism initiating contraction in VSMC. AngII induces a pharmacomechanical excitation–contraction coupling that occurs without changes of the membrane potential. 44 Several studies have suggested that microtubules can affect the contractile process in several cell types. 10,27 Despite this emerging evidence, the mechanisms of these observations have not been established, and the consequence of the alteration in response to AngII induced by isoflurane on VSMC contraction remains unknown. However, it may be linked to the role of AngII in short-term control of blood pressure during isoflurane anesthesia. 4

Another main result of this study is the lower sensitivity of the SHR to isoflurane as compared with the WKY strain. This difference cannot be linked to an interspecies variation in anesthetic potency, because anesthetic requirement of isoflurane was shown to be similar in WKY and SHR. 31 In contrast, it could be related to the structural and functional abnormalities of the arterial wall and to the alteration in Ca2+handling observed in VSMC from SHR. 18,20,23 Intracellular Ca2+concentration and Ca2+storage pools in cultured aortic VSMC from SHR were found to be increased during nonstimulated conditions. The response to various agonists, including AngII, is also enhanced in the SHR, as shown in the current study and in that by Cortes et al.19 In this regard, we observed an increase in the amplitude of intracellular Ca2+variation induced by AngII, suggesting an increase in Ca2+mobilization from internal stores in the SHR as compared with WKY, in accordance with previous studies. 17 Furthermore, regulation of AngII signaling pathways of the normotensive and hypertensive strains have been shown to differ in several respects, such as role of tyrosine kinase or mitogen-activated protein kinase, suggesting a signaling pattern characteristic of the hypertensive phenotype. 5,16,45 Whether this difference in sensitivity to isoflurane could be related to the greater instability of arterial blood pressure during isoflurane administration deserves further investigation. However, human hypertension differs in several points from hypertension of SHR, and our results cannot by directly extrapolated to humans with chronic hypertension.

Large conduit arteries do not contribute to peripheral vascular resistance, and the difference between the two strains we reported cannot be directly extrapolated to the regulation of arterial blood pressure during isoflurane anesthesia. However, Ca2+mobilization elicited by AngII was reported to differ only minimally between aortic and mesenteric vessels. 23 The model of aortic VSMC from SHR strain is of particular interest in hypertension, because the structural and functional changes of arterial wall have been linked to the alteration in arterial blood control and to detrimental consequences of hypertension. Furthermore, studying the effect of isoflurane on AngII-induced signaling pathways in this model could contribute to a better understanding of the molecular mechanisms implicated in the effect of anesthetics.

In conclusion, the current results established that isoflurane altered AngII-induced Ca2+mobilization from internal stores and Ca2+entry through capacitative Ca2+channels in cultured rat aortic VSMC. The hypertensive rats were less sensitive to isoflurane than normotensive ones. This effect of isoflurane was associated to a disorganization of the microtubular network and implicated similar mechanisms in both normontensive and hypertensive strains. These results may prove useful in understanding the alteration in vascular reactivity observed during isoflurane anesthesia.

Fig. 1. Representative recordings of the effect of 1 μm angiotensin II (AngII) on intracellular Ca2+increase in a control cell from WKY and in cells incubated in the presence of 1, 2, or 3% isoflurane. In protocols A
–D
, cells were exposed to AngII in the presence of external Ca2+(Cao2+). In protocols E
–H
, cells were incubated for 60 s in the nominal absence of external Ca2+and then exposed to Ang II. External Ca2+was reintroduced in the perfusion medium and AngII-induced Ca2+influx was estimated by the time course of Ca2+increase. In recordings B
–D
and F
–H
, cells were incubated in Na+–HEPES medium containing 1, 2, and 3% isoflurane, respectively (15 min). Ratios of the emission fluorescence (> 520 nm) measured at excitation wavelengths of 350 and 380 nm are shown on the ordinate.

Fig. 1. Representative recordings of the effect of 1 μm angiotensin II (AngII) on intracellular Ca2+increase in a control cell from WKY and in cells incubated in the presence of 1, 2, or 3% isoflurane. In protocols A
–D
, cells were exposed to AngII in the presence of external Ca2+(Cao2+). In protocols E
–H
, cells were incubated for 60 s in the nominal absence of external Ca2+and then exposed to Ang II. External Ca2+was reintroduced in the perfusion medium and AngII-induced Ca2+influx was estimated by the time course of Ca2+increase. In recordings B
–D
and F
–H
, cells were incubated in Na+–HEPES medium containing 1, 2, and 3% isoflurane, respectively (15 min). Ratios of the emission fluorescence (> 520 nm) measured at excitation wavelengths of 350 and 380 nm are shown on the ordinate.